New chemical biopsy tool for spatially resolved profiling of human brain tissue in vivo.

It is extremely challenging to perform chemical analyses of the brain, particularly in humans, due to the restricted access to this organ. Imaging techniques are the primary approach used in clinical practice, but they only provide limited information about brain chemistry. Solid-phase microextraction (SPME) has been presented recently as a chemical biopsy tool for the study of animal brains. The current work demonstrates for the first time the use of SPME for the spatially resolved sampling of the human brain in vivo. Specially designed multi-probe sampling device was used to simultaneously extract metabolites from the white and grey matter of patients undergoing brain tumor biopsies. Samples were collected by inserting the probes along the planned trajectory of the biopsy needle prior to the procedure, which was followed by metabolomic and lipidomic analyses. The results revealed that studied brain structures were predominantly composed of lipids, while the concentration and diversity of detected metabolites was higher in white than in grey matter. Although the small number of participants in this research precluded conclusions of a biological nature, the results highlight the advantages of the proposed SPME approach, as well as disadvantages that should be addressed in future studies.

Application across species of a one health approach to liquid sample handling for respiratory based -omics analysis.

Airway inflammation is highly prevalent in horses, with the majority of non-infectious cases being defined as equine asthma. Currently, cytological analysis of airway derived samples is the principal method of assessing lower airway inflammation. Samples can be obtained by tracheal wash (TW) or by lavage of the lower respiratory tract (bronchoalveolar lavage (BAL) fluid; BALF). Although BALF cytology carries significant diagnostic advantages over TW cytology for the diagnosis of equine asthma, sample acquisition is invasive, making it prohibitive for routine and sequential screening of airway health. However, recent technological advances in sample collection and processing have made it possible to determine whether a wider range of analyses might be applied to TW samples. Considering that TW samples are relatively simple to collect, minimally invasive and readily available in the horse, it was considered appropriate to investigate whether, equine tracheal secretions represent a rich source of cells and both transcriptomic and proteomic data. Similar approaches have already been applied to a comparable sample set in humans; namely, induced sputum. Sputum represents a readily available source of airway biofluids enriched in proteins, changes in the expression of which may reveal novel mechanisms in the pathogenesis of respiratory diseases, such as asthma and chronic obstructive pulmonary disease. The aim of this study was to establish a robust protocol to isolate macrophages, protein and RNA for molecular characterization of TW samples and demonstrate the applicability of sample handling to rodent and human pediatric bronchoalveolar lavage fluid isolates. TW samples provided a good quality and yield of both RNA and protein for downstream transcriptomic/proteomic analyses. The sample handling methodologies were successfully applicable to BALF for rodent and human research. TW samples represent a rich source of airway cells, and molecular analysis to facilitate and study airway inflammation, based on both transcriptomic and proteomic analysis. This study provides a necessary methodological platform for future transcriptomic and/or proteomic studies on equine lower respiratory tract secretions and BALF samples from humans and mice.

Breast Cancer: Targeting of Steroid Hormones in Cancerogenesis and Diagnostics.

Breast cancer is the most common malignancy in women with high mortality. Sensitive and specific methods for the detection, characterization and quantification of endogenous steroids in body fluids or tissues are needed for the diagnosis, treatment and prognosis of breast cancer and many other diseases. At present, non-invasive diagnostic methods are gaining more and more prominence, which enable a relatively fast and painless way of detecting many diseases. Metabolomics is a promising analytical method, the principle of which is the study and analysis of metabolites in biological material. It represents a comprehensive non-invasive diagnosis, which has a high potential for use in the diagnosis and prognosis of cancers, including breast cancer. This short review focuses on the targeted metabolomics of steroid hormones, which play an important role in the development and classification of breast cancer. The most commonly used diagnostic tool is the chromatographic method with mass spectrometry detection, which can simultaneously determine several steroid hormones and metabolites in one sample. This analytical procedure has a high potential in effective diagnosis of steroidogenesis disorders. Due to the association between steroidogenesis and breast cancer progression, steroid profiling is an important tool, as well as in monitoring disease progression, improving prognosis, and minimizing recurrence.

Investigation of steatosis profiles induced by pesticides using liver organ-on-chip model and omics analysis.

Several studies have reported a correlation between pesticides exposure and metabolic disorders. Dichlorodiphenyltrichloroethane (DDT) and permethrin (PMT), two pesticides highly prevalent in the environment, have been associated to dysregulation of liver lipids and glucose metabolisms and non-alcoholic fatty liver disease (NAFLD). However, the effects of DDT/PMT mixtures and mechanisms mediating their action remain unclear. Here, we used multi-omic to investigate the liver damage induced by DDT, PMT and their mixture in rat liver organ-on-chip. Organ-on-chip allow the reproduction of in vivo-like micro-environment. Two concentrations, 15 and 150 µM, were used to expose the hepatocytes for 24 h under perfusion. The transcriptome and metabolome analysis suggested a dose-dependent effect for all conditions, with a profile close to control for pesticides low-doses. The comparison between control and high-doses detected 266/24, 256/24 and 1349/30 genes/metabolites differentially expressed for DDT150, PMT150 and Mix150 (DDT150/PMT150). Transcriptome modulation reflected liver inflammation, steatosis, necrosis, PPAR signaling and fatty acid metabolism. The metabolome analysis highlighted common signature of three treatments including lipid and carbohydrates production, and a decrease in amino acids and krebs cycle intermediates. Our study illustrates the potential of organ-on-chip coupled to multi-omics for toxicological studies and provides new tools for chemical risk assessment.

Effect of the phenolic compounds from Sabara jaboticaba (Plinia jaboticaba (Vell. ) Berg) in reducing health risks caused by obesity / Efeito dos compostos fenólicos da jabuticaba Sabará (Plinia jaboticaba (Vell. ) Berg) na redução dos riscos à saúde causados pela obesidade

Sabara jaboticaba (Plinia jaboticaba (Vell.) Berg) is a Brazilian native fruit from Atlantic Forest, rich in polyphenols and appreciated for consumption both in natura and in various preparations. This study aimed to evaluate whether phenolic compounds of Sabara jaboticaba, in the form of phenolic extract (PEJ), can reduce the health risks caused by obesity and associated health problems induced by a fat-sucrose-rich diet (HFSD) in C57BL/6J mice. Initially, for 14 weeks, 66 8-week-old male mice were randomly distributed into two groups: negative control (CH), fed with standard AIN96M diet and water ad libitum; positive control (HFS), fed with HFSD and water ad libitum. At the end of this stage, 10 animals from each group were euthanized under anesthesia and their organs and tissues collected. The remaining animals were redistributed into four groups for another 14 weeks: group CH, fed a standard diet and water; HFS group, fed with HFSD and water; PEJ1 group, fed with HFSD and PEJ at the dose of 50 mg equivalent of gallic acid (GAE)/kg of body weight (BW); group J100 fed with HFSD and PEJ at the dose of 100 mg GAE/kg BW. Food intake, BW, and fasting blood glucose (FBG) were measured weekly and water (CH and HFS) or PEJ (PEJ1 and PEJ2) were daily administered. In the 26th week the intraperitoneal insulin tolerance test (ipITT) was performed, in the 27th, the oral glucose tolerance test (oGTT), and, in the 28th, the analyzes related to energy homeostasis. At the end of the experiment, the animals were euthanized under anesthesia and their organs and tissues were collected. When compared to the HFS group, animals that received PEJ showed decrease in BW gain of approx. 30% and of approx. 45% in the gain of total white adipose tissues (WAT). In addition, the PEJ groups showed less hypertrophied adipocytes. Inflammation markers were significantly reduced in both treated groups. The FBG was approx. 13% lower for the PEJ groups compared to the HFS group. In addition, the mean values of ipITT, oGTT, insulin and HOMA-IR demonstrated that PEJ increased insulin sensitivity and decreased glucose intolerance. GLUT4 expression in the muscle was also increased in the treated groups. The fecal lipid content was lower in the PEJ groups when compared to the HFS group, suggesting that PEJ inhibited pancreatic lipase activity both in vitro and in vivo. In the PEJ groups, the levels of total cholesterol, LDL and NEFA were reduced and those of HDL increased. The hepatic concentration of TAG was also reduced by PEJ. Energy expenditure and UCP1 expression were higher for both supplemented groups when compared to the HFS group. PEJ positively altered the intestinal microbiota and the analysis of metabolites showed that animals treated with PEJ had different metabolomic profile. Together, these results demonstrated that polyphenols from jaboticaba may be used as adjuvants against obesity and associated health problems

A jabuticaba Sabará (Plinia jaboticaba (Vell.) Berg) é um fruto nativo da Mata Atlântica brasileira, rico em polifenóis e apreciado para o consumo tanto in natura quanto em preparos variados. O objetivo deste trabalho foi avaliar se compostos fenólicos da jabuticaba Sabará, na forma de extrato fenólico (PEJ), são capazes de reduzir os riscos à saúde causados pela obesidade e problemas de saúde associados induzidos por uma dieta rica em lipídios e sacarose (HFSD) em camundongos C57BL/6J. Inicialmente, durante 14 semanas, 66 animais machos com oito semanas de vida foram distribuídos aleatoriamente em dois grupos: controle negativo (CH), alimentado com dieta padrão AIN96M e água ad libitum; controle positivo (HFS), alimentado com HFSD e água ad libitum. Ao final desta etapa, 10 animais de cada grupo foram eutanasiados sob anestesia e seus órgãos e tecidos coletados. Os animais restantes foram redistribuídos em quatro grupos por mais 14 semanas: grupo CH, alimentado com dieta padrão e água; grupo HFS, alimentado com HFSD e água; grupo PEJ1, alimentado com HFSD e PEJ na dose de 50 mg equivalente de ácido gálico (EAG)/kg de massa corporal (m.c.); grupo J100 alimentado com HFSD e PEJ na dose de 100 mg EAG/kg m.c. O consumo de ração, a massa corporal e a glicemia de jejum (FBG) foram medidos semanalmente e as gavagens de água (CH e HFS) ou PEJ (PEJ1 e PEJ2) foram realizadas diariamente. Na 26ª semana foi realizado o teste intraperitoneal de tolerância à insulina (ipITT), na 27ª o teste oral de tolerância à glicose (oGTT) e na 28ª as análises relacionadas a homeostase energética. Ao final do experimento os animais foram eutanasiados sob anestesia e seus órgãos e tecidos coletados. Quando comparados ao grupo HFS, animais que receberam o PEJ apresentaram ganho de massa corporal aprox. 30% menor e aprox. 45% menos massa total de tecidos adiposos brancos (TAB). Além disso, os grupos PEJ apresentaram adipócitos menos hipertrofiados. Marcadores de inflamação foram significativamente reduzidos em ambos os grupos tratados. A FBG foi aprox. 13% inferior para os grupos PEJ em relação ao grupo HFS. Além disso, os valores médios de ipITT, oGTT, insulina e HOMA-IR demonstraram que o PEJ aumentou a sensibilidade à insulina e diminuiu a intolerância à glicose. A expressão do GLUT4 no músculo estava aumentada nos grupos tratados. O conteúdo lipídico fecal dos grupos PEJ foi superior ao do grupo HFS, sugerindo que, assim como ocorreu in vitro, o extrato inibiu a atividade da lipase pancreática in vivo. Os níveis de colesterol total (PEJ1), LDL e NEFA foram reduzidos e os de HDL aumentados. A concentração hepática de TAG também foi reduzida pelo PEJ. O gasto energético e a expressão de UCP1 foram superiores para ambos os grupos suplementados quando comparados ao grupo HFS. PEJ alterou positivamente a microbiota intestinal e a análise de metabólitos mostrou que os animais tratados com PEJ possuíam perfil metabolômico diferente. Em conjunto, estes resultados demonstraram que os CFJS podem ser usados como adjuvantes no combate a obesidade e problemas de saúde associados

Sub-nanoliter metabolomics via mass spectrometry to characterize volume-limited samples.

The human metabolome provides a window into the mechanisms and biomarkers of various diseases. However, because of limited availability, many sample types are still difficult to study by metabolomic analyses. Here, we present a mass spectrometry (MS)-based metabolomics strategy that only consumes sub-nanoliter sample volumes. The approach consists of combining a customized metabolomics workflow with a pulsed MS ion generation method, known as triboelectric nanogenerator inductive nanoelectrospray ionization (TENGi nanoESI) MS. Samples tested with this approach include exhaled breath condensate collected from cystic fibrosis patients as well as in vitro-cultured human mesenchymal stromal cells. Both test samples are only available in minimum amounts. Experiments show that picoliter-volume spray pulses suffice to generate high-quality spectral fingerprints, which increase the information density produced per unit sample volume. This TENGi nanoESI strategy has the potential to fill in the gap in metabolomics where liquid chromatography-MS-based analyses cannot be applied. Our method opens up avenues for future investigations into understanding metabolic changes caused by diseases or external stimuli.

Hepatoxicity mechanism of cantharidin-induced liver LO2 cells by LC-MS metabolomics combined traditional approaches.

Hepatotoxicity induced by Mylabris has been reported in both clinical and animal experiments. Cantharidin (CTD), the main active compound of Mylabris was responsible for the hepatotoxicity, which aroused widespread concern. However, the mechanism of CTD hepatotoxicity remained unclear. In this study, LO2 cells were exposed to two doses of CTD (6.25 and 25 µM) for 12 h, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured. The metabolites in LO2 cells were profiled by LC-MS. Partial least squares discriminant analysis and orthogonal partial least squares discriminant analysis were used for screening potential biomarkers. The MetPA software was used for clustering and pathway analysis. Network pharmacology was used to predict the genes acted with potential biomarkers. Compared with the control group, the levels of ALT, AST, and LDH was significantly increased after CTD treatment. A total of 46 potential biomarkers for hepatotoxicity induced by CTD were identified. And downregulated potential biomarkers reflected the inhibitory effects of CTD toxicity on metabolism of LO2. Moreover, CTD-induced liver toxicity of LO2 cells is mainly related to three pathways: cysteine and methionine metabolism; glutathione metabolism; and glycine, serine, and threonine metabolism. Furtherly, the mRNA expression of CES2, DNMT1, NOS1, NOS3, S1PR2, and CES1 screened by network pharmacology were regulated by CTD. These studies provide valuable mechanistic insights into CTD-associated hepatotoxicity that will aid in the development of therapeutic prevention and treatment options for this liver disease.

Development of Pico-ESI-MS for Single-Cell Metabolomics Analysis.

In this chapter, we introduced a Pico-ESI strategy for metabolomics analysis with picoliter-level samples. This Pico-ESI strategy was technically achieved by pulsed direct current electrospray ionization source (Pulsed-DC-ESI). This source could collect MS signals for a few minutes from a cell, enabling us to obtain large-scale MS2 data of metabolite IDs in single-cell analysis. Further identification of the single-cell metabolome such as the database match and chemical modification to metabolome was thereby achieved. Technically, this source could ionize sample with no need of sample and electrode contact, which can be potentially applied for high-throughput analysis. We also introduced several strategies related to Pico-ESI to reduce the matrix interference especially for extremely small samples developed in our group, including step-voltage nanoelectrospray, picoliter sample desalting method, droplet-based microextraction method, and probe-ESI, etc. All these strategies have been successfully applied to single-cell analysis.

Comparison of underivatized silica and zwitterionic sulfobetaine hydrophilic interaction liquid chromatography stationary phases for global metabolomics of human plasma.

To increase metabolome coverage in global LC-MS metabolomics, often both reversed-phase liquid chromatography (RPLC) and hydrophilic-interaction liquid chromatography (HILIC) are implemented in parallel. However, there is a lack of consensus in the literature on the best HILIC stationary phase to employ for global metabolomics of human biological fluids. The objective of this study was to compare in detail the performance of two commonly employed HILIC phases: zwitterionic sulfobetaine ZIC-HILIC stationary phase and an underivatized silica HILIC stationary phase. During method development, the effect of salt concentration in the mobile phase was also investigated, and 5 mM ammonium acetate was selected. The stationary phases were evaluated using a mixture of 37 polar standards covering a range of logP values (-10 to 3.73), molecular weights (59-776 Da), charges (15 anions, 11 cations, and 11 neutral) as well as 17 lipid standards to understand phospholipid behaviour on the two stationary phases. The criteria used for the comparison included the quality of the chromatographic peak shape, adequate analyte retention, peak separation capability, and metabolite coverage. The zwitterionic ZIC-HILIC column provided better chromatographic performance over the silica stationary phase with 14 standards achieving good quality peaks compared to the 7 with the silica column. Only 2 standards were undetected with the ZIC-HILIC column compared to the 14 undetected with the silica column. In human plasma, 1966 and 1650 metabolites were observed on the ZIC-HILIC column in positive and negative electrospray ionization (ESI) respectively. On the silica HILIC column, 1773 and 2028 metabolites were observed in positive and negative ESI respectively, showing comparable performance of the two phases. Next, the effect of adding 10 mM ammonium phosphate to the samples to improve the analyte peak shape and metabolite coverage was investigated for both ZIC-HILIC and silica HILIC. In contrast with recently reported results for pZIC-HILIC, there was no clear evidence that ammonium phosphate addition was beneficial for human plasma samples. In conclusion, ZIC-HILIC provided better chromatographic performance for polar plasma metabolomics than underivatized silica in terms of chromatographic peak shape and chromatographic resolution, while maintaining comparable metabolite coverage. The addition of ammonium phosphate to human plasma was not beneficial for either of the two stationary phases.

Urinary metabolomics for discovering metabolic biomarkers of laryngeal cancer using UPLC-QTOF/MS.

The incidence of laryngeal cancer (LYC) is second only to lung cancer, which is also the second most common cancer in head and neck cancer. Risk assessment metabolomics biomarkers to diagnose LYC have not been found by now. To profile the metabolites in healthy controls (HCs) and LYC patients (LYCs), urine metabonomics study was performed based on reversed phase liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (RPLC-QTOF/MS). Six urine differentially expressed metabolites (Variable Importance in Projection >1, p < 0.05) were identified by searching reference library or comparing with standard based on OPLS-DA (orthogonal partial least squares-discriminant analysis) model. d-pantothenic acid, palmitic acid, myristic acid, oleamide, sphinganine and phytosphingosine were identified as differential metabolites associated with the LYC and they might play roles in sphingolipid metabolism, fatty acid biosynthesis, fatty acid elongation in mitochondria, pantothenate and coenzyme A (CoA) biosynthesis, beta-Alanine metabolism and fatty acid metabolism. These six differential metabolites were combined to test the potentiality of diagnosis of LYC. Results revealed that the area under the curve (AUC) value, sensitivity and specificity of receiving operator characteristic (ROC) curve were 0.97, 95% and 97%, respectively, indicating that this diagnosis method could be used to distinguish LYCs from HCs with good sensitivity and specificity.

Identification of novel serum biomarker for the detection of acute myeloid leukemia based on liquid chromatography-mass spectrometry.

Acute myeloid leukemia (AML) is a life-threatening hematological malignancy. Traditional diagnosis of AML depends on invasive bone marrow biopsies. To recognize the metabolic characteristics related with AML and search for early non-invasive biomarkers of AML, in this work we applied ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOFMS)-based metabolomoc method to profile serum metabolites from 55 de novo AML patients and 45 age- and gender-matched healthy subjects and to screen and validate AML biomarkers. We observed AML-related metabolic differences mainly involved in alanine, aspartate and glutamate metabolism; d-Glutamine and d-glutamate metabolism; tryptophan metabolism; taurine and hypotaurine metabolism; and phenylalanine metabolism as well as fatty acid metabolism. A serum metabolite biomarker panel consisting of glutamic acid, kynurenine and oleic acid was defined and validated based on binary logistic regression analysis and receiver operating characteristic curves (ROC) analysis, yielding an area under the ROC curve (AUC) of 0.981 with 0.975 sensitivity and 0.933 specificity in the discovery set and an AUC of 0.973 with 0.933 sensitivity and 0.933 specificity in the validation set. This work demonstrated the UHPLC- MS-based metabolomics as a low invasive potential tool for the detection of AML, and this composite serum metabolite panel exhibited good diagnostic performance for AML in this case-control study and deserved further validation in a large-scale clinical trial. The identified metabolic pathways were also potentially worthy of further studying the pathogenesis of AML.

Evaluating the Metabolic Alterations in Pancreatic Cancer.

Metabolic reprograming is an established hallmark of cancer cells. Pancreatic cancer cells, by virtue of the underlying oncogenic drivers, demonstrate metabolic reprograming to sustain growth, invasiveness, and therapy resistance. The increased demands of the growing tumor cells alter the metabolic and signaling pathways to meet the growing nutrient requirements. Investigating the metabolic vulnerabilities of tumor cells can help in developing effective therapeutics to target pancreatic cancer. In this chapter, we explain in detail the methods to evaluate the metabolic changes occurring in the tumor. This includes the glucose/glutamine uptake assays and the measurement of reactive oxygen species, extracellular acidification rate, and oxygen consumption rate in the tumor cells. All these physiological assays help in understanding the metabolic nature of the tumor.

Altered profiles and metabolism of l- and d-amino acids in cultured human breast cancer cells vs. non-tumorigenic human breast epithelial cells.

Herein we describe for the first time the endogenous levels of free l-and d-amino acids in cultured human breast cancer cells (MCF-7) and non-tumorigenic human breast epithelial cells (MCF-10A). d-Asp and d-Ser, which are co-agonists of the N-methyl-d-aspartate (NMDA) receptors, showed significantly elevated levels in MCF-7 cancer cells compared to MCF-10A cells. This may result from upregulated enzymatic racemases. Possible roles of these d-amino acids in promoting breast cancer proliferation by regulating NMDA receptors were indicated. d-Asn may also be able to serve as exchange currency, like specific l-amino acids, for the required uptake of essential amino acids and other low abundance nonessential amino acids which were elevated nearly 60 fold in cancer cells. The relative levels of specific l- and d-amino acids can be used as malignancy indicators (MIs) for the breast cancer cell line in this study. High MIs (>50) result from the increased demands of specific essential amino acids. Very low MIs (<1) result from the increased demands of specific d-amino acids (i.e., d-Ser, d-Asp) or the cellular release of amino acid exchange currency (i.e., l- and d-Asn) used in the upregulated amino acid antiporters to promote cancer cell proliferation.

13C Tracer Analysis and Metabolomics in 3D Cultured Cancer Cells.

Metabolomics and 13C tracer analysis are state-of-the-art techniques that allow determining the concentration of metabolites and the activity of metabolic pathways, respectively. Three dimensional (3D) cultures of cancer cells constitute an enriched in vitro environment that can be used to assay anchorage-independent growth, spheroid formation, and extracellular matrix production by (cancer) cells. Here, we describe how to perform metabolomics and 13C tracer analysis in 3D cultures of cancer cells.

Measuring In Vivo Tissue Metabolism Using 13C Glucose Infusions in Mice.

Metabolic alterations are a hallmark of cancer. While determining metabolic changes in vitro has delivered valuable insight into the metabolism of cancer cells, it emerges that determining the in vivo metabolism adds an additional layer of information. Here, we therefore describe how to measure the in vivo metabolism of cancer tissue using 13C glucose infusions in mice.

Measuring Human Lipid Metabolism Using Deuterium Labeling: In Vivo and In Vitro Protocols.

Stable isotopes are powerful tools for tracing the metabolic fate of molecules in the human body. In this chapter, we focus on the use of deuterium (2H), a stable isotope of hydrogen, in the study of human lipid metabolism within the liver in vivo in humans and in vitro using hepatocyte cellular models. The measurement of de novo lipogenesis (DNL) will be focussed on, as the synthesis of fatty acids, specifically palmitate, has been gathering momentum as being implicated in cellular dysfunction, which may be involved in the development of non-alcoholic fatty liver disease (NAFLD). Therefore, this chapter focusses specifically on the use of 2H2O (heavy water) to measure hepatic DNL.

Measuring Rates of ATP Synthesis.

Here, we offer you a high-throughput assay to measure the ATP synthesis capacity in cells or isolated mitochondria. More specifically, the assay is linked to the mitochondrial' electron transport chain components of your interest being either through complex I (with or without a linkage to pyruvate dehydrogenase activity), through complex II, or through the electron transport flavoprotein and complex I (ß-oxidation of fatty acids).

Determining the Impact of Metabolic Nutrients on Autophagy.

Tumorigenesis relies on the ability of cancer cells to obtain necessary nutrients and fulfill increased energy demands associated with rapid proliferation. However, as a result of increased metabolite consumption and poor vascularization, most cancer cells must survive in a nutrient poor and high cellular stress microenvironment. Cancer cells undergo metabolic reprogramming to evade cell death and ensure proliferation; in particular, cancer cells utilize the catabolic process of autophagy. Autophagy creates an intracellular pool of metabolites by sequestering cytosolic macromolecules in double-membrane vesicles targeted for lysosomal degradation. During times of environmental stress and nutrient starvation, autophagy is upregulated through the dynamic interactions between two nutrient sensing proteins, AMP activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), in cooperation with Unc-51 like autophagy activating kinase 1 (ULK1). In this way, a lack of metabolic nutrients plays a critical role in inducing autophagy, while the products of autophagy also serve as readily available fuel for the cell. In this chapter, we describe methods to visualize and quantify autophagy using a fluorescent sensor of autophagic membranes. Thus, the impact of specific nutrients on autophagy can be measured using live-cell fluorescent microscopy.

Measuring the Activation of Cell Death Pathways upon Inhibition of Metabolism.

Nutrient starvation or inhibition of cellular metabolism can induce cancer cell death. This can be measured by a variety of methods. We describe here four simple methods to measure cell death in culture by using microscopy, western blot, and flow cytometry. We also provide tools to differentiate between different forms of cell death like apoptosis and necrosis by using chemical inhibitors.